Engaging Students and Teachers as Community Scientists in DNA Barcoding Initiatives.

Historically, contributions to scientific knowledge have been perceived as something that only professional scientists have the ability to affect. This has led to the belief that scientific pursuits are done not by everyday people but by individuals who have no connection to the communities that their discoveries might impact. DNA barcoding initiatives have the potential to bridge this gap. Community leaders, students, teachers, and other community members can come together with engaged scientists to solve relevant issues that affect them. Over the last 20 years, DNA barcoding has been used successfully in a variety of educational contexts to incorporate original research into school curricula and informal outreach and education programs. DNA barcoding is especially suitable for educational settings because it is conceptually and technically straightforward, the workflow is adaptable to a variety of situations, and free and open-access online tools exist that allow participants to contribute high-quality data to international research efforts. DNA barcoding also offers a unique service-learning opportunity, where participants gain both knowledge and confidence in science. This is important because a growing body of evidence suggests that actively conducting research increases student and teacher engagement and retention of students in science. Here, we describe a framework and case studies in different educational settings that can be modeled and adapted to various educational contexts.

Depalmitoylated Ras traffics to and from the Golgi complex via a nonvesicular pathway.

Palmitoylation is postulated to regulate Ras signaling by modulating its intracellular trafficking and membrane microenvironment. The mechanisms by which palmitoylation contributes to these events are poorly understood. Here, we show that dynamic turnover of palmitate regulates the intracellular trafficking of HRas and NRas to and from the Golgi complex by shifting the protein between vesicular and nonvesicular modes of transport. A combination of time-lapse microscopy and photobleaching techniques reveal that in the absence of palmitoylation, GFP-tagged HRas and NRas undergo rapid exchange between the cytosol and ER/Golgi membranes, and that wild-type GFP-HRas and GFP-NRas are recycled to the Golgi complex by a nonvesicular mechanism. Our findings support a model where palmitoylation kinetically traps Ras on membranes, enabling the protein to undergo vesicular transport. We propose that a cycle of depalmitoylation and repalmitoylation regulates the time course and sites of Ras signaling by allowing the protein to be released from the cell surface and rapidly redistributed to intracellular membranes.

Postprenylation CAAX processing is required for proper localization of Ras but not Rho GTPases.

The CAAX motif at the C terminus of most monomeric GTPases is required for membrane targeting because it signals for a series of three posttranslational modifications that include isoprenylation, endoproteolytic release of the C-terminal- AAX amino acids, and carboxyl methylation of the newly exposed isoprenylcysteine. The individual contributions of these modifications to protein trafficking and function are unknown. To address this issue, we performed a series of experiments with mouse embryonic fibroblasts (MEFs) lacking Rce1 (responsible for removal of the -AAX sequence) or Icmt (responsible for carboxyl methylation of the isoprenylcysteine). In MEFs lacking Rce1 or Icmt, farnesylated Ras proteins were mislocalized. In contrast, the intracellular localizations of geranylgeranylated Rho GTPases were not perturbed. Consistent with the latter finding, RhoGDI binding and actin remodeling were normal in Rce1- and Icmt-deficient cells. Swapping geranylgeranylation for farnesylation on Ras proteins or vice versa on Rho proteins reversed the differential sensitivities to Rce1 and Icmt deficiency. These results suggest that postprenylation CAAX processing is required for proper localization of farnesylated Ras but not geranygeranylated Rho proteins.

Topology of mammalian isoprenylcysteine carboxyl methyltransferase determined in live cells with a fluorescent probe.

Isoprenylcysteine carboxyl methyltransferase (Icmt) is a highly conserved enzyme that methyl esterifies the alpha carboxyl group of prenylated proteins including Ras and related GTPases. Methyl esterification neutralizes the negative charge of the prenylcysteine and thereby increases membrane affinity. Icmt is an integral membrane protein restricted to the endoplasmic reticulum (ER). The Saccharomyces cerevisiae ortholog, Ste14p, traverses the ER membrane six times. We used a novel fluorescent reporter to map the topology of human Icmt in living cells. Our results indicate that Icmt traverses the ER membrane eight times, with both N and C termini disposed toward the cytosol and with a helix-turn-helix structure comprising transmembrane (TM) segments 7 and 8. Several conserved amino acids that map to cytoplasmic portions of the enzyme are critical for full enzymatic activity. Mammalian Icmt has an N-terminal extension consisting of two TM segments not found in Ste14p and therefore likely to be regulatory. Icmt is a target for anticancer drug discovery, and these data may facilitate efforts to develop small-molecule inhibitors.

Thematic review series: lipid posttranslational modifications. CAAX modification and membrane targeting of Ras.

Proteins that terminate with a consensus sequence known as CAAX undergo a series of posttranslational modifications that include polyisoprenylation, endoproteolysis, and carboxyl methylation. These modifications render otherwise hydrophilic proteins hydrophobic at their C termini such that they associate with membranes. Whereas prenylation occurs in the cytosol, postprenylation processing is accomplished on the cytoplasmic surface of the endoplasmic reticulum and Golgi apparatus. Among the numerous CAAX proteins encoded in mammalian genomes are many signaling molecules such as monomeric GTPases, including the Ras proteins that play an important role in cancer. In the course of their processing, nascent Ras proteins traffic from their site of synthesis in the cytosol to the endomembrane and then out to the plasma membrane (PM) by at least two pathways. Recently, retrograde pathways have been discovered that deliver mature Ras from the PM back to the Golgi. The Golgi has been identified as a platform upon which Ras can signal. Thus, the subcellular trafficking of Ras proteins has the potential to increase the complexity of Ras signaling by adding a spatial dimension. The complexity of Ras trafficking also affords a wider array of potential targets for the discovery of drugs that might inhibit tumors by interfering with Ras trafficking.

PKC regulates a farnesyl-electrostatic switch on K-Ras that promotes its association with Bcl-XL on mitochondria and induces apoptosis.

K-Ras associates with the plasma membrane (PM) through farnesylation that functions in conjunction with an adjacent polybasic sequence. We show that phosphorylation by protein kinase C (PKC) of S181 within the polybasic region promotes rapid dissociation of K-Ras from the PM and association with intracellular membranes, including the outer membrane of mitochondria where phospho-K-Ras interacts with Bcl-XL. PKC agonists promote apoptosis of cells transformed with oncogenic K-Ras in a S181-dependent manner. K-Ras with a phosphomimetic residue at position 181 induces apoptosis via a pathway that requires Bcl-XL. The PKC agonist bryostatin-1 inhibited the growth in vitro and in vivo of cells transformed with oncogenic K-Ras in a S181-dependent fashion. These data demonstrate that the location and function of K-Ras are regulated directly by PKC and suggest an approach to therapy of K-Ras-dependent tumors with agents that stimulate phosphorylation of S181.